The increase in average life expectancy implies an overload in tissues and organs demand. In the last few years a great variety of hydrogels—class of three-dimensional, highly hydrated polymeric networks (water content ≧30% of total weigh)—has been developed and applied in tissue regeneration. These materials are composed of hydrophilic polymer chains, which can be either synthetic or natural, appealing for tissue engineering strategies due to the possibility of reproducibly mimetizing the chemical structure of biological tissues and its properties in general.
Synthetic and natural polymers have been explored as drug carriers. Unfortunately, the majority of the polymers used clinically, although well tolerated, are still non-biodegradable synthetic polymers e.g. poly(ethyleneglycol) (PEG) (Fuertges and Abuchowski, 1990) and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, (Vasey et al., 1999). Thus in order to ensure renal elimination and to exclude the threat of progressive accumulation after repeated administration, only polymers with a molecular weight below the renal threshold (approximately 40,000 Da) can be used (Hreczuk-hirst et al., 2001).
A variety of natural materials may be used to form hydrogels for tissue engineering, such as collagen, chitosan, alginate and hyaluronic acid (HA). However, the performance of these materials in vivo is not always the best, as stated by Drury and Mooney (2003). Highly pure and homogeneous chitosan is quite difficult to produce; on the other hand, the pro-inflammatory bioactivity limits its biomedical usage. The safety of collagen materials is a concern, due to the risk of contamination. Alginate is also several times highlighted as a promising polysaccharide for hydrogels synthesis for tissue engineering applications. However, alginate is not specifically degraded, undergoing a slow uncontrolled dissolution, therefore being difficulty cleared of the body. Additionally, polymers like pullulan (Nogusa et al., 1995) or dextran (Nishikawa et al., 1996), also referred in this area, being inherently biodegradable, even with low levels of functionalization (to promote a drug linkage or to decrease degradation rates) can easily become non-degradable (Vercauteren et al., 1996). Some of these naturally derived polymers (including dextran) are immunogenic, impeding its repeated administration (Hreczuk-hirst et al., 2001). A Phase I study involving dextran-doxorubicin has shown evidences of hepatotoxicity (Danauser-Reidl et al., 1993).
Injectability is commonly a target property when developing hydrogels, assuring its administration in a minimally invasive procedure. Crosslinking time is a very important feature of these materials, determining its suitability to a specific application. For instance, a gelling period greater than 30 min is not adequate to a maxillofacial intervention.
The present invention, aiming to surpass the setbacks mentioned above, describes an injectable dextrin-based hydrogel, with potential for inclusion and transportation of biomolecules, drugs, nanogels, and granular compounds, as well as for cell encapsulation. Simultaneously, this biomaterial is intended to provide controllable crosslinking times and mechanical properties, through the adjustment of specific parameters, such as the degree of oxidation and concentration of the crosslinking agent. Dextrin means a glucose polymer produced by hydrolysis of starch, consisting of glucose units linked mainly by α-1,4 linkages. In addition to α-1,4, there may be a proportion of α-1,6 linkages, the amount depending on the source of the starch. Any dextrin is a mixture of glucose polymers of different chain lengths.
Dextrin-based hydrogel formulations are referred in a small number of articles and patents, reviewed ahead. Indeed, dextrin is an emergent tool in the biomedical field for its non-toxicity and non-immunogenicity (Treetharnmathurot et al., 2009). It has been FDA approved as the peritoneal dialysis solution Icodextrin™. Icodextrin (a polydisperse dextrin) has also been developed as carrier solution for intraperitoneal administration of an anticarcinogenic agent (Kerr et al., 1996). Recent work also reported the ability of dextrin conjugates to exhibit anti-endotoxin activity as well as to regulate the inflammatory response (Davtyan et al., 2007; Avetisyan et al., 2006). In another recent work, dextrin-hydroxyapatite (HAp) complex was used as a bone filling material, with good performance (Asai et al., 2009). In addition, a nanogel, organized by self-assembling of amphiphilic dextrin has been described as potential drug carriers (Gonçalves et al., 2007). A similar material—a dextrin nanogel—has been developed also by Orienti et al. (2009; WO2009/016663A1). Dextrin-based microspheres were used for encapsulation of the photosensitizer porphyrin, which aggregates in aqueous solutions, allowing its administration in the monomeric form, in photodynamic therapy (Luz et al., 2008). Colin Brown (2010) developed also a dextrin formulation capable of preventing or reducing the incidence on postoperative adhesions (US2010/0240607A1).
Recently, Carvalho et al. (2007) produced dextrin hydrogels, namely dextrin-VA and dextrin-HEMA, as controlled drug delivery systems. However, the method used to produce these hydrogels, i.e. radical polymerization, requires chemical initiators to activate the gelification process (e.g. ammonium persulfate), which might react with cellular structures and reveal toxic. In addition, it implies the modification of the dextrin main chain with acrylic monomers, and the gelification is fast. Hence, these dextrin-based materials do not possess the main required properties for an injectable hydrogel, essentially due to limitations such as cytotoxicity and lack of control over crosslinking times.
The proven clinical tolerability of dextrin, readily degraded by amylases (Davies, 1994), suggests it might gather excellent properties for the development of drug carrier systems and overall in biomedical applications. For this purpose it is particularly relevant the fact that dextrin is an abundant resource, being already available in a medical-grade formulation with excellent biocompatibility. Additionally, long plasma circulation times (hours or days) has been achieved by functionalization of the main polymeric chain, allowing an improved ability for tissue targeting (Hreczuk-hirst et al., 2001; Hardwicke et al., 2008; Treetharnmathurot et al., 2009). Dextrin's low molecular weight is also a crucial and determinant property, once it favors a healthy renal clearance.
The U.S. Pat. No. 5,541,234, by Unger et al., describes hydrogels with high porosity and low density, made of alginate and/or other polysaccharides, including dextrin, in which the polymer concentration originating the ideal density for the porous structure lies preferably between 1% and 10%. In the particular cases of agar, carrageenan, gelatins and caseins the crosslinking process takes place preferably at high temperatures, turning in situ gelation unfeasible, otherwise tissues surrounding the hydrogel could be seriously damaged. It is also stated the use of solvents along the crosslinking reaction, which, bearing in mind biomedical applications, is a clear disadvantage compared to the process described in the present invention. Unger et al. do not ply the possibility of associating biomolecules or cells. Moreover, the gelification relies strictly on the use of crosslinking agents, without previous modification of the polysaccharides. Dextrin, a glucose polymer, needs a pretreatment, e.g., oxidation by periodic acid, as proposed in this invention, allowing for the posterior gelification by the addition of a reticulating agent. Furthermore, still in the scope of the present invention, the polymer concentration that guarantees the ideal texture is 30%, giving rise to an injectable hydrogel, with appealing crosslinking times (5-30 min), which allows its unhurried handling and implantation, when used in maxillofacial surgery applications, as an adjuvant to osteogenic granular compounds. A polymer concentration bellow 25% will originate a viscous fluid instead of an hydrogel.
Bouhadir et al. (1999; US2007/07186413) conceived hydrogels for the controlled release of pharmaceuticals, based on the use of polysaccharides crosslinked with adipic acid dihydrazide (ADH), as in the present invention. The preferred polysaccharide is alginate. The procedure leading to the production of a hydrogel includes the partial oxidation of alginate followed by polymerization with ADH. This way, through oxidation of the polysaccharide, the authors aim the production of a material degradable in vivo. Indeed, the non-degradability is a main limitation to the biomedical use of alginates, since it avoids its efficient elimination and excretion. A better control over the gelification reaction is also envisaged, since the ionotropic gelification commonly carried out with alginate is unsatisfactory. In still another aspect of the patent, drug-polymer conjugates are developed, allowing an improved control of the drug release, in this case not based just on mass transfer phenomena.
In the case of the present invention, the retention of the biomaterial in the kidneys is not an issue, due to the low molecular weight of dextrin, whose degradation and removal may be controlled through the degree of substitution. In this regard, dextrin brings a clear advantage with when compared to alginates. On the other hand, in the present invention a strategy to avoid the quick release of pharmaceuticals from the highly porous hydrogel is purposed. Indeed, an additional degree of control on drug release may be achieved using nanogels with hydrophobic cores, able to solubilize poorly water-soluble pharmaceuticals, simultaneously allowing an additional control over the pharmacokinetic properties of the system. This is a rather simpler approach than the strategy purposed by Bouhadir and colleagues, based on the use of pharmaceutical-polymer conjugates. Additionally, these authors do not provide information on the profile of degradation of the hydrogels in physiologic medium, nor regarding its porosity, crucial parameters concerning its viability as drug controlled release systems. Furthermore, the alginate hydrogels reveal comparatively poor mechanical properties. For the same concentration of crosslinking agent (ADH), the resistance of the hydrogel to compressive forces (proportional to the number of intermolecular bonds) is lower for the alginate hydrogel, which is likely to translate also a poorer biodegradation profile.
In the U.S. Pat. No. 6,991,652 B2, Burg and colleagues describe composites made of a porous matrix of microparticles with variable geometry (spheres, cylinders or a net), preferentially made of collagen, which may be carried in a liquid or viscous fluid. In this invention, the composites are cultivated with cells, which may proliferate and originate a neo-tissue. Furthermore, the composites may be administrated by injection, in a minimally invasive manner, being claimed to be useful for a wide range of tissue engineering applications. Dextrin is referred as one of the materials which may constitute the fluid phase which carries the porous matrix, however the patent does not describe how hydrogels may be obtained from dextrin, whose function seems to be to increase the viscosity of the fluid phase. In the present invention, dextrin hydrogels may be associated e.g. to bioactive granules, ceramic particles, biomolecules or cells, as in the invention by Burg et al., and also with nanogels, namely dextrin nanogels, endowing the composite material with improved versatility, namely allowing its use for the transport and controlled release of bioactive molecules. Furthermore, this invention includes a methodology for the gelification of dextrin, which results in controllable mechanical properties and biodegradability, favoring its use for the controlled release of pharmaceuticals associated with the transport of a solid phase in an injectable system.
The patent WO2005/042048A2, from Hill et al., published in 2005, describes the production of injectable hydrogels made of proteins and polysaccharides, with gelification times of about 2 hours, allowing the incorporation of pharmaceuticals, namely, but not only, for bone regeneration. The concept developed for the gelification strategy is in this case based on the reactivity of the amine groups of the proteins and the polysaccharides. In the case of neutral polysaccharides the oxidation must be carried out first. This way, a lengthy gelification process results, as opposed to the obtained in the case of the present invention through the use of ADH.
The Japanese patent JP2005/298644A2, developed by Akiyoshi and colleagues, describe the production of a hybrid hydrogel, made of pullulan. The hydrogel is obtained by radical polymerization, using a mixture of methacryloil-pullulan with a nanogel of pullulan, also methacrylated. The nanogel self-assembles through hydrophobic interaction of cholesterol moieties grafted on the polysaccharide. This way, a hydrogel with a dispersed nanogel able to carry biopharmaceuticals, namely proteins, is obtained. The present invention also contemplates the incorporation of a nanogel as a drug controlled release device, differing from the Akiyoshi invention in relevant aspects, namely the high molecular weight of the pullulan used by Akiyoshi et al. (100 kDa) (as opposed to the low molecular weight dextrin) and the use of a polymerization initiator, 2,2′-Azobis [2-(2-imidazoline-2-yl) propane. While the patent does not describe details regarding the injectability of the hydrogel, the high molecular weight of pullulan raises doubts concerning the efficiency of the biological excretion, even more because it is not clear whether the intermolecular bonds are degradable. As a matter of fact, as it has been reported for methacryloil-dextran hydrogels, it is likely that methacryloil-pullulan hydrogels are not degradable in vivo (Cadée et al., 2000). On the other hand, the initiator may be toxic (Ameer et al., 2001). Thus, the use of the dextrin hydrogel, as well as of the dextrin nanogel, associated with the gelification method based on oxidized dextrin and ADH as reticulating agent, offer significant advantages, regarding biocompatibility and excretability. Later, Akiyoshi et al. (JP2009/149526A2) reported the use of the same hydrogel for the controlled release of cytokines.
The present invention is unique as it introduces a hydrogel made of dextrin, a hydrogel obtained through chemically simple and inexpensive methods, without using toxic initiators or catalysts. These processes originates a convenient speed of gelification as for handling the material, allowing its injectability into e.g. a tissue defect, for regeneration purposes, allowing the production of hydrogels with suitable mechanical properties and biodegradability for each application. These characteristics make possible the easy incorporation e.g. of biomolecules, bioactive ceramics and cells, as well as of nanogels (for instance of dextrin), resulting, in the late case, in a multidimensional composite (with a hydrophobic phase dispersed at the nano level), with improved versatility in the perspective of its use as a carrier for the controlled release of bioactive molecules.
The present invention relates to the production of hydrogels made of dextrin and adipic acid dihydrazide, which can be injectable, with application as 1) scaffold for tissue regeneration; 2) as a carrier of bioactive microspheres, e.g. bioactive osteogenic granular compounds for bone regeneration adjuvancy; 3) cell encapsulation; 4) vehicle for bioactive molecules (namely proteins or polysaccharides, e.g. collagen) which promote cell adhesion and proliferation, and 5) self assembled nanogels (e.g. made of dextrin) associated drug delivery systems.
In this invention, an expeditious methodology is used to prepare degradable hydrogels from oxidized dextrin (oDex) and adipic acid dihydrazide, without the use of any chemical initiator. Gelation periods from 1 to 30 minutes can be obtained, depending on the components concentration.
Dextrin homopolysaccharide chain can be oxidized using periodic acid. The periodate ion attacks one of the hydroxyl groups of the vicinal diol in dextrin residues, between C2-C3 positions of the glucopyranoside ring, breaking the C—C bond and yielding two reactive aldehyde groups. Aldehydes react with molecules such as adipic acid (ADH), which in turn acts as a reticulating agent, giving rise to hydrogels. The concentration of crosslinker directly affects the density of intermolecular bonds, which in turn influences the mechanical, degradation ang gelling properties of hydrogels. However, the amount of ADH used must be optimized taking into account the number of available oxidized residues, so that the number of viable chemical bonds is maximized, in detriment of the occurrence of pendant groups. Furthermore, the presence of excessive reactive groups may compromise the mechanical properties of hydrogels by effects of steric hindrance, for which the degree of functionalization of the polymeric chain must be moderate.
oDex/ADH hydrogels are degradable both hydrolytically, at the level of its covalent intermolecular bonds, and enzymatically, through the action α-amylase. Depending on the crosslinking density it is possible to obtain different degradation profiles. The tight structure of highly interconnected hundred nanometer pores becomes gently loose along the degradation process, allowing to predict some compliance towards cell invasion, once the material is injected, specially if the matrix exhibits chemoattractant signals or adhesion peptides, which may be easily introduced (for instance using nanogels loaded with bioactive molecules).
In terms of biocompatibility, dextrin hydrogels are non-toxic. Cells adhere and proliferate along its interface, allowing the perspectivation of a good tissue-hydrogel interaction in vivo. Additionally, oDex/ADH hydrogels are non-haemolytic.
Many advantages can be highlighted regarding oDex/ADH hydrogels in comparison with other known hydrogels, namely 1) simple and expeditious methodology of production; 2) absence of chemical initiators (generally toxic); 3) low molecular weight-favoring biodegradation and renal clearance; 4) biocompatibility; 5) possibility of inclusion/encapsulation of specific molecules/cells; 6) low-cost, naturally derived raw material, and already available in medical grade; 7) potential to perform as a carrier of microparticulate systems, for instance for bone regeneration applications; 8) great potential to perform as a controlled drug release system, both for biopharmaceuticals (therapeutic proteins) or water-insoluble molecules (through incorporation of nanogels with hydrophobic cores).